Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control

Abstract

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Figure 1. Rational design of peptides.

(A) X-ray crystal structure of the Dengue virus envelope protein (1OAN.pdb). (B) Active fragment of the virus fusion peptide, VG16. (C) The amino acid sequences of the designed peptides VG16A and VG16KRKP. (D) LPS (1 EU/ml) neutralization and corresponding MIC values (in μM) against Gram-positive and Gram-negative bacteria and fungi for the peptides used in this study.

Figure 2. Cell lysis by VG16KRKP.

(A) ¹H NMR spectrum of a solution of 1.5 mM VG16KRKP, 10 mM PBS, pH 7.2 in the absence (1) or in the presence of 10⁹ cells after 30 min (2), 3 h (3) or 9 h (4) of co-incubation. Spectral regions characterized by the appearance of new signals are highlited in green. (B) Changes in broadening and intensity of VG16KRKP resonances after cell addition and cell dilution. (1) ¹H NMR spectrum of a solution containing 1 mM VG16KRKP, 10 mM PBS, pH 7.2, 64 scans; (2) ¹H NMR spectrum of a solution containing 1 mM VG16KRKP and 10⁹ cells, 10 mM PBS, pH 7.2, 64 scans (2× in intensity); (3) ¹H NMR spectrum of a solution containing 1 mM VG16KRKP and 3.3×10⁹ cells, 10 mM PBS, pH 7.2, 64 scans (2× in intensity). The last sample was obtained by 1:3 dilution of the sample in 2 with a 1 mM VG16KRKP solution. (C–E) Chemical shift difference of VG16KRKP ¹H resonances after cell addition and cell dilution, evidenced by expansions of spectra depicted in panel D. (C) NεH resonace of Trp (spectrum 1, 4× intensity; spectra 2 and 3, 8× intensity); (D) aromatic resonances around 7 ppm (spectrum 1, 2× intensity; spectra 2 and 3, 4× intensity); (E) Hα and aliphatic region (spectrum 1, 1× intensity; spectra 2 and 3, 2× intensity). All spectra were acquired on 600 MHz at 25 °C. (F) SEM images of E. coli in the (i) absence and (ii) presence of 10 μM of VG16KRKP. (G) SEM images of X. oryzae in the (i) absence and (ii) presence of 10 μM of VG16KRKP. All images were taken 45 min post incubation at 25× magnification.

Figure 3. Binding studies of VG16KRKP with LPS.

(A) Intrinsic tryptophan fluorescence emission spectra of VG16A or VG16KRKP in the absence and presence of LPS micelle. (B) Upper panel showing endothermic heat of reaction vs. time (minute) upon interaction of VG16KRKP with LPS micelle. The lower panel shows enthalpy change per mole of peptide injection vs. molar ratio (peptide:LPS) for VG16KRKP. In this experiment, 50 μM of LPS micelle was titrated against 200 μM of peptide, VG16KRKP. TEM images of LPS micelle (C) alone and (D) in the presence of VG16KRKP. Scale bar = 1 μm. (E) ³¹P NMR spectra of the phosphates of LPS micelle in presence of MnCl₂ in free form and upon addition of increasing concentrations of VG16KRKP signifying the fragmentation of LPS micelles, as evident from the reduction in intensity.

Figure 4. Structure of VG16KRKP in LPS.

Selected regions of two-dimensional ¹H-¹H trNOESY spectra of VG16KRKP in LPS showing (A) fingerprint region of C^αH-NH resonances, and (B) long-range NOEs between aromatic ring protons and aliphatic side chain residues. Peaks, which are marked by the symbol * are unassigned due to the cis-trans configuration at Cys9-Pro10 bond. All experiments were performed on a Bruker Avance 500 MHz at 25 °C. (C) Side chain representation of twenty-ensemble VG16KRKP structures in LPS. (D) Cartoon representation of average structure of VG16KRKP conformations bound to LPS. The hydrogen bonds, which help in the stabilization of structures, are shown as black dotted lines. All the positive charges are facing one side, marked by circle. (E) Hydrophobic packing constituted by the residues Val1, Ala2, Trp5, Pro10, Leu11 and Phe12 are shown by space filling. Interestingly, the positive charge residues maintain a distance similar to the distance between two phosphate head groups of LPS (~12 Å). Depth of insertion study using fluorescence quencing experiments show that the position of Trp5 residue is ~6.8 Å from the center of the LPS bilayer, suggesting the Trp and othe residues of VG16KRKP, associated with Trp have strong van-der-Waals interaction with the acyl chain of LPS. (F) Superposition of DPC bound fusion domain of hemagglutinin (1IBN.pdb) (structure is stabilized by i, i + 5 residues) and LPS bound VG16KRKP (2 MWL.pdb) (structure is stabilized by i, i + 7 residues).

Figure 5. Hemolytic and cytotoxic effects of VG16KRKP.

(A) Bar plot showing percentage of hemolysis of human RBC upon addition of increasing concentrations of VG16KRKP. (B) Bar plot showing percentage viability of human fibrosarcoma cell line upon treatment with increasing concentrations of VG16KRKP.

Figure 6. Application of VG16KRKP in treating X. oryzae infection in rice plants.

(A) Images of 67 days old uprooted control, infected, and peptide-treated rice plants showing inhibition of disease upon treatment. (B) Bar plots showing variation in wet weight of plants (i), healthy leaves (ii), root length (iii), and shoot height (iv) between control, infected and peptide-treated rice plants. A student’s t-test was performed in each case. (C) Bar plot of number of colony forming units (CFU) of X. oryzae obtained from equal amounts of crushed leaf tissue of control, infected, and peptide-treated plants which show a 10-fold reduction in the number of colonies in treated plants, confirming that VG16KRKP is capable of inhibiting disease.